Oligonucleotides for the detection of salmonella

ABSTRACT

The subject of the invention is new means, comprising nucleotide sequences, for the detection, especially after amplification, of the DNA or of the cDNA of S. enterica or S. bongori. The invention relates especially to the oligonucleotides represented in the abstract figure.

This application is a continuation of application Ser. No. 08/586,272,filed on Jan. 16, 1996, now U.S. Pat. No. 5,824,795.

BACKGROUND OF THE INVENTION Description of the Related Art

The genus Salmonella contains two species, Salmonella enterica, aspecies divided into six sub-species on the basis of biochemicalcharacteristics and those of homologies at the DNA level, and Salmonellabongori. The genus is subdivided into more than 2000 serovarietiesdefined with the aid of somatic and flagellar antigens. Bacteria of thegenus Salmonella are generally pathogenic for animals and for man. It isthus known that Salmonella is among the agents responsible for the mostcommon cases of food poisoning in developed countries; that is why rapidand reliable methods for the detection of subspecies of Salmonella areimportant.

The salmonellae responsible for food toxi-infections belongpredominantly to the subspecies I (also called group I) of S. enterica.

Toxi-infections are however not the only pathologies caused bySalmonella infections.

For example, Salmonella enterica subspecies enterica serovariety typhi(called hereinafter Typhi) is the causative agent of human typhoidfever.

Given the nature of the infections caused by salmonellae and the needespecially to search for their presence in biological samples taken frompatients or from foods, it appears essential to have available rapid andsensitive means for detecting their presence therein.

The standard culture methods widely used up until now for the detectionof salmonellae require a substantial amount of time and are not suitablefor example for monitoring the contamination of food products. In orderto overcome the disadvantages of these methods, several methods based onmolecular biology techniques such as hybridization tests and tests basedon the polymerase chain reaction have already been proposed. Various DNAprobes have been used in several hybridization and PCR procedures todetect the Salmonella subspecies in the diet. However, none of thesetechniques is completely satisfactory since the sequences used are notcompletely known or are not exclusively present in the genus Salmonellaand thus can lead to cross-reactions between the probe and DNA sequencesfrom other enterobacteria or can lead to a large number of falsenegatives or false positives.

The inventors have sought means allowing the specific and sensitivedetection of all the salmonellae of the species S. enterica and/or S.bongori. To this end, they focused their attention on the strainSalmonella enterica subspecies enterica serovariety typhi (S. Typhi) andon the gene involved in the invasion of cells by S. Typhi.

Furthermore, they defined certain conditions allowing the specificdetection of defined groups of Salmonellae, for example Group Ibacteria.

It has already been shown in the prior state of the art that the Typhistrain is capable of adhering to monolayers of HeLa cells and ofentering into these cells (Yabuuchi et al, 1986). However, up until now,the genetic determinants involved in this process of adhering to andentering into the cells have not been clearly identified. Elsinghorst etal (1989) have cloned a Typhi chromosomal fragment which confers onEscherichia coli type bacteria the capacity to penetrate into Henle 407cells. Recently, another chromosomal region involved in the invasion ofHeLa cells by the Typhi Ty2 strain was identified and cloned (Popoff andDion, 1990).

SUMMARY OF THE INVENTION

The inventors of the present application have identified on an S. typhiDNA fragment of 2.4 kb, contained in the HindIII sequence of 7.9 kbdescribed by Popoff and Dion (1990), regions capable of taking part inthe activity of invading Salmonella enterica subspecies entericaserovariety Typhi in cells, and in particular in HeLa type cellcultures, these regions being capable, in addition, of being used inreactions for carrying out a generalized diagnosis of all therepresentatives of the species S. enterica and/or S. bongori oroptionally under special detection conditions, for the specificdiagnosis of S. enterica group I.

A sequence called IagA and a sequence called IagB have been identifiedby the inventors and characterized as taking part in the cell invasionwhich manifests itself during an infection due to Salmonella entericasubspecies enterica serovariety Typhi.

The specificity of these sequences within S. Typhi has led the inventorsto propose their use in order to define means for the diagnosis of aninfection by S. typhi or even for the diagnosis of an infection bySalmonella of the species S. enterica and/or S. bongori or in certaincases for the detection of S. enterica of specific groups.

These means, which can be used for the diagnosis of an infection bySalmonella enterica and/or Salmonella bongori comprise oligonucleotidescapable of being used in reactions for the amplification of nucleotidesequences, for example polymerase chain reactions. The invention alsorelates to probes for the detection of nucleic acids of S. enterica orof a specific subspecies of S. enterica and/or of S. bongori, thesenucleic acids being, where appropriate, amplified fragments.

The subject of the invention is also a kit and a method for detectingthe presence of Salmonella enterica and/or of Salmonella bongori inbiological samples and for example in food products or in any samplewhich is the subject of a clinical diagnosis. These detection kits andmethods are, according to a specific embodiment of the invention,specific for the group I strains of S. enterica.

According to another embodiment of the invention, these methods make itpossible, on the contrary, to search for the presence of S. enterica orS. bongori bacteria of the genus Salmonella. The genus Salmonella thuscomprises six subspecies or groups I, II, III, IV, V or VI. Thesubspecies I, II, III, IV and VI belong to the species S. enterica andthe subspecies V belongs to the species S. bongori.

The invention also relates to the nucleotide sequences taking part inthe invasion of cells by Salmonella enterica subspecies entericaserovariety Typhi, characterized in that they are one of the sequencesiagA or iagB respectively between nucleotides 97 and 1755 of thesequence represented in FIG. 1 (IagA) and between nucleotides 1776 and2255 of the sequence represented in FIG. 1 (IagB) (SEQ ID NO: 1-3).

The invention also relates to nucleotide sequences which are modified inrelation to iagA or iagB but exhibit nevertheless the same properties asregards the invasion of cells, or hybridize under stringent conditionswith one of the abovementioned sequences.

The subject of the present application is also IagA and IagB proteinscorresponding to the sequences presented in FIG. 1 or variants of thesesequences which are obtained by mutation, deletion or addition of aminoacids, as long as the sequence thus obtained is recognized by antibodiesdirected against one of the abovementioned IagA or IagB sequences (SEQID NO: 2-3).

In general, the subject of the invention is any amino acid sequenceencoded by the iagA and iagB genes represented in FIG. 1.

The invention relates, moreover, to any fragment especially any purifiedfragment of one of these sequences which is sufficient to allow S. typhito preserve its properties of adhering and infecting cells, inparticular HeLa cells in culture.

The process for infecting HeLa cells in culture is the standard processwhich has been especially described in the international patentapplication published under number WO 92/01056.

According to another aspect, the invention relates to means fordetecting the presence of S. enterica and/or S. bongori and, whereappropriate, for quantifying S. enterica and/or S. bongori in biologicalsamples.

Biological sample is understood to mean any sample collected forcarrying out in vitro analyses in animals or in man or collected fromfood products regardless of their nature or from any liquid, solid orgaseous medium likely to contain the desired pathogenic agents.

The subject of the invention in this context is a nucleotide sequencecomprising at least 9 nucleotides, characterized in that it hybridizeswith one of the IagA or IagB sequences presented above.

The hybridization conditions referred to above are defined according tothe desired specificity of the hybridization and appropriate conditionsare given as a guide in the examples of the present application.

Preferably, the invention relates to oligonucleotides derived from theC-terminal part of the iagA sequence represented in FIG. 1 (SEQ ID NO:1-2).

Oligonucleotide type sequences can be selected for use as primers eitherfor the detection, after amplification, of the genomic DNA or the cDNAof Salmonella of the species S. enterica and/or of the species S.bongori belonging to the other groups I to VI or part of these groups,or under other conditions for the specific detection of S. entericagroup I. These may be especially nucleotide sequences obtained bychemical synthesis according to methods known to a person skilled in theart.

Preferred oligonucleotides which can be used for the amplification ofnucleic acid characteristic of bacteria belonging to one of the groupsI, II, IIIa, IIIb, IV, V or VI of the genus Salmonella and especially ofthe genomic DNA or the cDNA of S. enterica and/or of S. bongori, are forexample the following (their position within the IagA sequencerepresented in FIG. 1 being indicated) (SEQ ID NO: 4-14):

position

Iag1: 5'-TA TTA AGT ATG CAG GTT ATG-3' 1424-1443

Iag2: 5'-AGA GAA TTT CTG CAA AGT GAA-3' 1585-1605

Iag3: 5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT-3' 1495-1521

Iag4: 5'-GAG CGT GCC TTA CCG ACG ATA-3' 1564-1584

Iag5: 5'-GCA GGG ATC ACT AAG CTG TG-3' 1318-1337

Iag6: 5'-CGT GGG CAA CCA GCA CTA ACG-3' 1637-1657

Slm1: 5'-CG GGT TAA AGG TTA TCA CCT-3' 709-728

Slm2: 5'-AG CAT GGC GCA AAT GGG-3' 1014-1031

Slm3: 5'-GCA CCA GGA AAG CAT TAA GTT GAT AGA ACA C-3' 732-762

Slm4: 5'-CTT CGC TGG GAC ACA AAG CA-3' 823-842

SS28: 5'-TAA TGC TTT CCT GGT GC-3'.

Other oligonucleotides capable of being used as primers for theamplification of DNA or of cDNA from the iagB gene of all Salmonellastrains of the species S. enterica and/or of S. bongori have beendefined from the iagB sequence represented in FIG. 1.

The subject of the invention is therefore the oligonucleotidescorresponding to the following chains (SEQ ID NO: 15-18):

Iag7: 5'-T ACG GCA TGG GCT GAT TGC T-3'

Iag8: 5'-T TAC GCT ATC GCC CAG CAG CAG GA-3'

Iag9: 5'-T GGT CAT AAC CGA GAT GGT TCA AAC GAT C-3'

Iag10: 5'-A CAG TTG TTA CAG GAT CCC T-3'

These oligonucleotides can also be used as probes, for example for thedetection of the products of amplification of the DNA and/or of the cDNAof S. enterica and/or of S bongori.

A pair of primers which is preferred for carrying out the amplificationof the nucleic acid of S. enterica and/or of S. bongori, regardless ofthe group to which the bacterium belongs, is for example composed of theprimers Iag5 (sense) and Iag6 (anti-sense).

This pair of primers direct the amplification of a 340 bp nucleic acidfragment.

Another preferred pair of primers is composed of the primers Slm1(sense) and Slm2 (antisense). These primers are capable of hybridizingwith the DNA or the cDNA of S. enterica and/or of S. bongori bacteria ofone of the groups I, II, III, IV, V or VI.

According to another preferred embodiment, the invention relates to theoligonucleotides which can be used as primers for the specific detectionof Salmonella enterica Group I when the detection conditions after theamplification of the DNA or of the cDNA are those which are described inexample I.

Such primers are characterized by their capacity to amplify nucleic acidsequences from S. enterica or S. bongori strains representative of thegroups I, II, III, IV, V and VI, but for which the detection conditionsare those set out in example I allowing the detection of the group Ibacteria alone.

A pair of oligonucleotides which can be used for these purposes, asprimers specific for the detection of DNA or cDNA sequences from S.enterica group I for example consists of the following sequences (SEQ IDNO: 19, 14):

SS2 5'-CCGGGCAGATGATACCC-3' and

SS28'-TAATGCTTTCCTGGTGC-3'.

The oligonucleotides defined by the inventors make it possible toenvisage the diagnosis of S. enterica and/or of S. bongori undersatisfactory conditions in terms of sensitivity speed, ease andspecificity.

The subject of the invention is also a kit for the detection of S.enterica and/or of S. bongori by amplification of the genomic orcomplementary DNA of S. enterica and/or of S. bongori, characterized inthat it comprises:

oligonucleotides as described above, which are capable of hybridizingunder stringent conditions with the genomic DNA or the cDNA of S.enterica and/or of S. bongori,

a probe for the detection of the amplified fragments corresponding toone of the definitions given in the preceding pages,

the reagents necessary for carrying out the amplification reaction.

The subject of the invention is therefore in particular the use of theabovementioned oligonucleotides as primers for the amplification of aDNA or cDNA sequence from Salmonella enterica and/or from Salmonellabongori, which is contained in one of the iagA or iagB sequences asdescribed in the preceding pages or which are complementary to such asequence, or alternatively the use of these oligonucleotides as probefor the detection of an amplified nucleotide sequence.

For example, the oligonucleotides iag5 and iag6 can be used respectivelyas sense and antisense primers for the detection of S. enterica and/orof S. bongori of the group I, II, III, IV, V or VI.

Likewise, the pair of primers Slm1 and Slm2 can be used for thedetection of bacteria of the species S. enterica and/or S. bongori fromone of these groups in a biological sample.

The invention also relates to the use of the oligonucleotides SS2 andSS28 for the specific detection in vitro, in a biological sample, of S.enterica group I.

The detection is specific when the primers used for the amplification ofthe desired nucleotide sequences allow the amplification of S. entericaand/or S. bongori bacteria belonging to one of the other groups II, III,IV, V or VI, but that the conditions used do not allow the detection ofbacteria of these same groups or of different organisms which arecapable of being present in the biological sample tested.

The invention thus relates to a set of oligonucleotides which can beused for the detection of S. enterica and/or S. bongori bacteria afteramplification of the genomic or complementary DNA of S. enterica and/orof S. bongori, characterized in that it comprises:

a pair of oligonucleotides corresponding to the definitions given above,which are capable of hybridizing under stringent conditions with thegenomic DNA or the cDNA of S. enterica and/or of S. bongori,

a probe corresponding to the characteristics given above.

A first set of oligonucleotides which can be used for the in vitrodetection, in a biological sample, of Salmonella enterica and/or S.bongori strains belonging to one of the groups I, II, III, IV, V or VI,is characterized in that it contains the following oligonucleotides (SEQID NO: 8-9):

the sequence Iag5 (5'-GCA GGG ATC ACT AAG CTG TG-3' and the sequenceIag6 (5'-CGT GGG CAA CCA GCA CTA ACG-3') which can be used as primersfor the amplification and

the sequence Iag3 (5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT-3') which canbe used as revealing probe and the sequence Iag4 (5'-GAG CGT GCC TTA CCGACG ATA-3') which can be used as capture probe (SEQ ID NO: 6-7).

Another set of oligonucleotides which can be used for the specificdetection in vitro, in a biological sample, of S. enterica group I, ischaracterized in that it comprises the following oligonucleotides (SEQID NO: 17-15):

SS2 (5'-CCGGGCAGATGATACCC-3' and

SS28 ('-TAATCGTTTCCTGGTGC-3').

The subject of the present application is moreover an iagA proteinencoded by the nucleotide sequence iagA represented in FIG. 1, as wellas a protein iagB encoded by the nucleotide sequence iagB represented inFIG. 1.

Preferably, the iagA and iagB proteins have respectively the amino acidsequences represented in FIG. 1.

Also entering within the framework of the invention is a process for thein vitro detection, in a biological sample, of Salmonella entericaand/or S. bongori nucleotide sequences previously amplified for exampleby PCR, characterized in that it comprises the steps of:

denaturing the amplified S. enterica and/or S. bongori sequence,

bringing the denatured amplified nucleotide sequences from S. entericaand/or from S. bongori into contact with a capture probe and a revealingprobe which are obtained from the oligonucleotides defined above underconditions allowing the hybridization of the said capture and revealingprobes with the above-mentioned amplified nucleotide sequence from S.enterica and/or from S. bongori, the capture probe being attached to thesurface of a well of a microtitre plate and the revealing probe beinglabelled and free in an appropriate hybridization buffer;

incubating the reaction mixture for a sufficient time to allow thehybridization reaction;

washing in order to remove the unreacted oligonucleotides;

revealing the revealing probes having hybridized to the amplifiednucleotide sequences.

The detection process described above may be advantageouslycharacterized in that the detection is carried out in accordance withthe following steps:

denaturation of a 10 μl volume of the amplified sequence by addition,volume for volume, of a 200 mM NaOH, 40 mM EDTA solution,

prehybridization of the microplates whose well surface is coated withthe capture probe, in an appropriate hybridization buffer,

release of the microplate and filling of each of the wells with 200 μlof hybridization buffer containing the denatured amplified fragment andthe revealing probe labelled with peroxidase at the concentration of 10ng/μl,

incubation of the mixture for one hour at 37° C., with stirring,

washing of the mixture which has reacted with a 10 X washing solution(100 mM Tris, 3 M NaCl, 1% Tween 20, pH 7.4),

detection of the activity of the peroxidase linked to the probe bycolorimetry in the presence of a colored substrate.

The revealing of the activity of the peroxidase present on the revealingprobe can be obtained by carrying out the following steps:

deposition of 200 μl of a 40 mM trisodium citrate solution, 0.03% H₂ O30%, 7.5 mg/ml of orthophenylenediamine (OPD) in each of the wellscontaining the reaction mixture,

incubation of the microplate for 30 min in the dark and at 37° C.,

blocking of the reaction by addition of 50 μl/well of a 4 N H₂ SO₄solution,

determination of the optical density at a wavelength of 492 nm(reference at 620 nm).

Advantageously, the capture probe used is the oligonucleotide Iag4 andthe revealing probe is the oligonucleotide Iag3.

Thus, the means defined within the framework of the invention allow thequalitative or quantitative detection of the presence of S. entericaand/or S. bongori type bacteria, whether this is a nonspecific detectionwithin one of the groups I, II, III, IV, V or VI of S. enterica and/orof S. bongori.

Under specific conditions for carrying out the detection step, as setout in example I, the primers SS2, SS28 and the probe SS40 allow on thecontrary the specific detection of S. enterica group I bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages of the invention appear in thefollowing examples and in the figures:

FIG. 1: Nucleotide sequence of a 2.4 kb DNA fragment of the invasionregion of Salmonella ser. Typhi. The potential sites for binding to theribosome are underlined (SEQ ID NO: 1-3).

FIG. 2: Percentage of the activity obtained with various strains ofSalmonella belonging to various serovarieties by sandwich hybridization.Serovarieties of various Salmonella isolates tested:

a: S. enterica subspecies enterica (1), ref.: C53

b: S. enterica subspecies salamae (II), ref.: 975-71

c: S. enterica subspecies salamae (II), ref.: 3975-83

d: S. enterica subspecies arizonae (IIIa), ref.: 1600 K

e: S. enterica subspecies arizonae (IIIa), ref.: So 20--20

f: S. enterica subspecies diarizonae (II), (ref.: 5250-85

g: S. enterica subspecies diarizonae (IIIb), ref.: 8013-93

h: S. enterica subspecies houtenae (IV), ref.: 1357-73

i: S. bongori, ref.: 2790-79

k: S. enterica subspecies indica (VI), ref.: 4355-84-7, 6, 5, 4 and 3:log (amount of DNA molecules).

FIG. 3: Alignment of the sequences of the amplified fragments(nucleotides 1345 to 1644) of the 6 groups of Salmonellae (SEQ ID NO:20-27).

FIG. 4: Amplification by means of the primers Iag5 and Iag6 on tworepresentatives of each of the groups of Salmonellae.

FIG. 5: Autoradiography of the Southern blot of the amplified productsof Salmonellae.

FIG. 6: Determination of the minimum number of chromosomal DNA moleculeswhich can be detected. Autoradiography of the Southern blot andmicroplate hybridization.

FIG. 7: Location of the oligonucleotides selected within the IagA gene.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE I CLONING ANDSEQUENCING OF THE 2.4 kb DNA FRAGMENT

This DNA fragment was subcloned using a restriction fragment obtained bycutting with the HindIII enzymes, from the 7.9 kb HindIII sequencedescribed in the publication by Popoff and Dion, 1990, into derivativesof the vector m13 (Messing and Vieira, 1982).

After carrying out this cloning, the chain termination dideoxy methodwas carried out using the modified T7 DNA polymerase (Sequenase, USBCorp.) and universal synthetic oligonucleotides as primers. All the endsof the restriction fragments used overlap with each other. Thesequencing of the DNA was carried out at least twice on each of thestrands. The nucleotide sequence was analyzed using the Lipan andPearson programme, 1985.

As shown by the sequence presented in FIG. 1, two open reading framesare contained in the fragment sequenced; they are designated by theterms iagA (abbreviation for invasion associate gene) and iagB. The twoopen reading frames are transcribed in the same orientation. The firstATG codon (bp 97) of the open reading frame of iagA which is preceded bythe sequence 5'-AGAGA-3' is supposed to correspond to the site ofinitiation of translation of the iagA gene. The iagA gene encodes apolypeptide containing 553 amino acid residues with a calculatedmolecular weight of 63026 Da. A significant homology was detectedbetween the N-terminal domain of the IagA protein and the domaincorresponding to the protein for regulation of transcription PhoB (24%identity and 52% similarity for a superposition of 108 amino acids) andthe protein PhoP (25% identity and 69% similarity for 100 aligned aminoacids) of E. coli. The ATG codon for initiation of the iagB gene (bp1776) is also preceded by a potential ribosome-binding site(5'-AGGAAG-3'). The iagB gene encodes a polypeptide containing 160 aminoacids and having a calculated molecular weight of 18369 Da. Comparisonof the sequence of the IagB protein with the translated sequencescontained in the Genbank databank has shown a significant homology withthe protein IpgF (43% identity and 66% similarity for 151 aligned aminoacids).

The IpgF protein is encoded by the ipgF gene which is situated on theplasmid associated with the virulence of Shigella flexneri, at the 5'end of the mxi-spa locus (Allaoui et al, 1993).

The Salmonella enterica subspecies enterica serovariety Typhi proteinsdetected are therefore thought to have a role in the infection by thesebacteria, and especially in the adhesion and the penetration into thecells.

EXAMPLE 2 SPECIFIC DETECTION OF S. ENTERICA GROUP I

A procedure for detecting the subspecies of Salmonella by the polymerasechain reaction (PCR) has been developed. A pair of oligonucleotides usedas primer was defined in order to amplify a 93 bp fragment of a generequired for the invasion of HeLa cells by S. typhi, strain Ty2. Theamplification product was analyzed by a nonradioactive sandwichhybridization on microtitre plates using two different oligonucleotidesaccording to the procedure described by Chevrier et al, 1993, Mol. Cell.Probes 7, 187-197. The capture oligonucleotide was phosphorylated at its5' end and covalently linked to wells carrying amine-containing groupsof a microtitre plate. The detection oligonucleotide was aminated at its5' end and then labelled with a biotinyl-N-hydroxy-succinimide ester.After hybridization, the hybrid molecules were detected by avidinconjugated to alkaline phosphatase and to a chromogenic substrate. Thismethod requires the use of only a thermal cycler and a conventionalmicrotitre reader, and can be carried out on a large scale.

MATERIALS AND METHODS Bacterial Strains

Two hundred and twenty-eight clinical isolates (Table 1) including S.bongori (Sambrook et al, 1989, Molecular cloning, a laboratory manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), S.enterica subspecies I(116), II(56), IIIa(11), IIIb(30), IV(5) and VI(5)and 16 non-salmonella Enterobacteria strains (Table 2) representing 9different genera were used in this study. The S. ser. Typhimurium C53strain was used as positive control, and the E coli HB101 strain wasused as negative control in the PCR tests.

Extraction of DNA

The strains were cultured in an LB medium at 37° C. In order to carryout the rapid extraction of the DNA, 2 ml of the culture maintainedovernight were centrifuged and resuspended in 1 ml of TE (10 mM Tris-HClbuffer at pH 8 containing 1 mM EDTA). The cells were centrifuged, thecentrifugation pellet was resuspended in 500 μl of sterile distilledwater and heated at 100° C. for 10 minutes. Finally, the solution wascentrifuged and the supernatant was stored for the PCR experiments.

Oligonucleotide Primers and Probes

The oligonucleotides were synthesized in a cyclone DNA synthesizer(Millipore-Waters) using the phosphoramidite technology.

The sequences of the oligonucleotide primers were the following (SEQ IDNO: 14, 19):

SS2: 5'-CCGGGCAGATGATACCC-3' and

SS28: 5'-TAATGCTTTCCTGGTGC-3'.

The capture oligonucleotide probe,

SS40: 5'-CCCGAACTATCTCGATCTGTACAATATTATCATT-3'

was phosphorylated at its 5' end with T4 polynucleotide kinase(Boehringer) according to the description made by Sambrook et al, 1989,(Molecular cloning, a laboratory manual. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) (SEQ ID NO: 28). The octadecanucleotidedetection probe SS41 (5'-GCAGGTGATAACCTTTAA-3') was synthesized with anamino functional group at its 5' end using the solid phasephosphoramidite method in an Applied Biosystem 380B DNA synthesizer andthen labelled with D-biotinyl-Σ-aminocaproic acid-N-hydroxysuccinimideester (Boehringer) according to the description made by Tham et al,1990, (FEMS Microbiol. Lett. 69, 109-116) (SEQ ID NO: 29). The captureand detection oligonucleotides were both purified on a rapid HR 10/10desalting column with the FPLC system (Pharmacia).

PCR Experiments

The DNA fragment subjected to the PCR reaction was denatured directly inthe wells by adding sequentially 95 μl of distilled water, 5 μl of PCRsample, 40 μl of detection probe and 14 μl of 1 N NaOH per well. After10 minutes, neutralization was carried out by adding 21 μl of 1 M NaH₂PO₄ containing 1% sarkosyl. All the samples were prepared in duplicate.After the neutralization, the band was deposited on a metallic surfaceand maintained in an oven overnight at 40° C. The final concentration ofthe biotinylated detection probe SS41 was 0.5 nM. During the incubationin the oven, it is preferable not to leave the unused wells empty but tofill them with water so as to obtain homogeneous thermal exchanges. Themicrowells were washed 5 times at room temperature with TBS-Tw (0.15 MNaCl, 10 mM Tris-HCl buffer at pH 8, 1% Tween 20). 100 μl of alkalinephosphatase-extravidine conjugate (Sigma), diluted at 1 μg/ml in TBS-Twcontaining 1% bovine serum albumin, were added per well. Next, the bandwas incubated at room temperature for 1 h, washed 5 times with TBS-Twand finally 200 μl of 1 M diethanolamine at pH 9.8 containing 1 mM MgCl₂and 1 mM para-nitrophenyl phosphate were added. The enzyme reaction wascarried out for 30 minutes to 2 hours. The absorbance was measured at405 nm using a microplate reader (Dynatech). The signal obtained withthe standard solution of the amplified DNA fragment (800 fm/well) of S.ser Typhimurium strain C53 was considered to represent 100% and used asreference for each hybridization test. The blank values correspond tothe mean absorbance measured in the wells coated with theoligonucleotide SS40 incubated with only 0.5 nM of biotinylatedoligonucleotide probe SS41.

RESULTS

Optimization of the Method

The primers and the probes were chosen in the iagA sequence. Variouspairs of primers were tested in order to optimize the sandwichhybridization technique on CovaLink microplates. The pair of primerschosen (SS2 and SS28) allowed the specific amplification of the 93 bpregion of the Salmonella genomic DNA. By using this pair of primers, itwas shown that a standard MgCl₂ concentration (1.5-2 mM) led to arelatively unadvantageous amplification result and that an MgCl₂concentration of 4 mM was necessary in order to obtain an efficientamplification. Internal oligonucleotides, SS40 and SS41, were used in anonradioactive hybridization test as capture probe and detection proberespectively.

Specificity of the Technique

The specificity of the method for the detection of salmonellae wasevaluated with 228 strains of Salmonella (Table 1) and 16 heterologousbacteria strains (Table 2). The results are summarized in Table 3.Edwardsiella tarda, Klebsiella pneumoniae, species of Enterobacter andAcinetobacter, Pasteurella, Vibrio harveyi, Serratia marcescens and moresubstantially species of Citrobacter and all E coli gave a hybridizationsignal of less than 20%. On the basis of this value, it was concludedthat all the Salmonella strains belonging to the subspecies I could bedetected by the present method. Furthermore, only one strain (strain3975-83) of the 56 strains of the subspecies II and 3 strains of the 11strains of the subspecies IIIa gave a positive signal. Salmonellabongori and the strains belonging to the subspecies IIIb, IV and VI werenot detectable.

Detection Level of the Technique with Whole Bacteria

1/10th dilutions of a suspension of the S. ser Typhimurium C53 strain(from 10⁹ to 10⁻² cells/ml) were made in order to estimate the minimumnumber of bacteria which could be detected by PCR followed by thenonradioactive hybridization technique. DNA was extracted from eachcalibrated suspension using the technique of rapid extraction byboiling. The results obtained show clearly that the technique of rapidextraction of DNA by simply boiling the suspension before the PCRreaction is an efficient technique. Indeed, it allows the detection ofonly one cfu unit.

                  TABLE 1                                                         ______________________________________                                        Salmonella subspecies used to evaluate the specificity                        of the DNA hybridization tests.                                                                    No. of  No. of                                           Microorganism tested isolates                                                                              serovars                                         ______________________________________                                        Salmonellia enterica subsp enterica I                                                              116     43                                               serovar Adelaide             1                                                Agona                        2                                                Altona                       1                                                Angoda                       1                                                Bardo                        2                                                Blockley                     1                                                Bovismorbificans             3                                                Braenderup                   4                                                Brandenburg                  1                                                Bredeney                     1                                                Broughton                    2                                                Cerro                        1                                                Chester                      1                                                Coeln                        1                                                Concord                      1                                                Dakar                        1                                                Derby                        2                                                Enteridis                    28                                               Georgia                      1                                                Hadar                        1                                                Heidelberg                   4                                                Ibadan                       2                                                Indiana                      1                                                Infantis                     5                                                Lexington                    1                                                London                       1                                                Mbandaka                     1                                                Montevideo                   6                                                Moscow                       1                                                Ohio                         1                                                Orion                        1                                                Panama                       3                                                Paratyphi B                  2                                                Saintpaul                    1                                                Typhimurium                  13                                               Typhisuis                    1                                                Vaertan                      1                                                Veneziana                    1                                                Vinohrady                    1                                                Virchow                      10                                               Wien                         1                                                Woodinville                  1                                                Yolo                         1                                                Salmonella enterica subsp salamae II                                                               56      56                                               Salmonella enterica subsp arizonae IIIa                                                            11      29                                               Salmonella enterica subsp diarizonae IIIb                                                          30      5                                                Salmanella enterica subsp houtenae IV                                                              5       5                                                Salmonella enterica subsp indica VI                                                                5       5                                                Salmonella bongori   5       5                                                (initially S. enterica subsp bongori V)                                       ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Heterologous bacteria used in the DNA hybridization                           test                                                                          Genus        Species     Number of isolates                                   ______________________________________                                        Escherichia  coli        4                                                    Edwarsiella  tarda       1                                                    Citrobacter  amalonaticus                                                                              1                                                                 freundii    1                                                    Klebsiella   pneumoniae  1                                                    Enterobacter agglomerans 1                                                                 asburiae    1                                                                 hormoechei  1                                                    Pasteurella  multocida   1                                                    Acinetobacter                                                                              lwoffii     1                                                                 haemolyticus                                                                              1                                                    Vibrio       harveyi     1                                                    Serratia     marcescens  1                                                    ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    Clinical strains of bacteria and controls tested in a Sandwich                hybridization test                                                            S. enterica                                                                              S. enterica                                                                        S. enterica                                                                        S. enterica                                                                        S. enterica                                                                        S. enterica     Control                        subsp.     subsp.                                                                             subsp.                                                                             subsp.                                                                             subsp.                                                                             subsp.    Non-  without                        enterica   salamae                                                                            arizonae                                                                           diarizonae                                                                         houtenae                                                                           indica                                                                             S. bongori                                                                         Salmonella                                                                          DNA                            __________________________________________________________________________    activity (%)                                                                  100%-20%                                                                            116   1   3     0   0    0    0    0     0                              19%    0   51   8    12   4    4    5    9     0                              > blank                                                                       < blank                                                                              0    4   0    18   1    1    0    7     23                             Total 116  56   11   30   5    5    5    16    23                             __________________________________________________________________________

Quantitative Hybridization with Purified Genomic DNA

The nonradioactive hybridization procedure used in the tests reportedhere can be easily carried out in quantitative studies. To compare thehybridization signals obtained with various Salmonella strains, DNA wasextracted from 10 strains representing the 6 subspecies of Salmonellaenterica and the species Salmonella bongori, then calibrated quantitiesof DNA were subjected to PCR reactions followed by a sandwichhybridization. The results are reported in FIG. 2. It was demonstratedthat the hybridization signal obtained with 10⁷ molecules of DNA ofSalmonella bongori or of the subspecies II, IIIa, IIIb, IV and VI ofSalmonella enterica is lower than the hybridization signal observed with10³ molecules of DNA of the strains of the subspecies I. However, it isimportant to note that the isolate 3975-83 (subspecies II) gave the samehybridization signal as the strains belonging to the subspecies I.

DISCUSSION

PCR amplification allows a very sensitive detection of specific DNAsequences. The sensitivity of the amplification depends essentially onthe number of copies of target DNA, the purity of the sample to beanalyzed, the DNA extraction method, and the sensitivity of the methodused to detect the PCR products. Visualization of the PCR products byethidium bromide staining in an electrophoresis gel is not compatiblewith the routine use of the technique and is not sufficiently sensitive.The sensitivity can be enhanced by the use of double PCR or of DNAprobes with a Dot-blot or a Southern-blot hybridization. However, doublePCR is very sensitive to contamination by DNA and the Dot-blot orSouthern-blot hybridization techniques are not appropriate forautomation. Microplate hybridization therefore offers an appropraitetechnique for the detection and quantification of fragments amplified byPCR. The simple covalent attachment of the nucleic acids to microwellsrepresents an advantageous variant of passive adsorption and asubstantial improvement for the detection of fragments amplified by PCRon micro-wells.

It is known that the strains of Salmonella which cause infections in manbelong essentially to the subspecies I. Indeed, more than 95% of theclinical isolates in humans belong to this subspecies (Rowe, B., 1987,Salmonella surveillance. Reports received from centers participating inthe WHO programme. World Health Organization London). Furthermore, in1991, the "Centre National d'Etudes Veterinaires et Alimentaires" ofParis (France) reported [Corbion, B. et al, 1991, Inventory ofSalmonella] that in the previous years, most of the strains isolated inanimals in the diet or in the environment in 1988 and 1989 (that is tosay, 18832 strains) belong to the subspecies I (99.2%).

The results reported here have made it possible to define a method basedon PCR amplification for the detection of pathogenic strains ofSalmonella. A pair of primers, SS2 and SS28, and a pair of probes, SS40and SS41 were selected from a gene necessary for the invasion of HeLacells by Salmonella ser. Typhi strain Ty2. By using the combination ofthe PCR technique and microplate nonradioactive sandwich hybridization,all the Salmonella bacteria of the subspecies I were detected.

The detection limit was lower than a threshold represented by 10 cellsper PCR tube, which is in accordance with the results obtained by othersimilar PCR techniques. Given the nucleic acid similarity betweenmembers of the enterobacteria, it was important to check the specificityof these new primers and probes with the enterobacteria genera which aremost likely to lead to "false-positive" type reactions. From the resultsobtained, it can be concluded that no false-positive reaction can takeplace when the PCR and hybridization conditions described above arefollowed.

It is advantageous to note that the Salmonella strain 3975-83(subspecies II) had a hybridization signal identical to that obtainedwith the isolates belonging to the subspecies I. This strain wasisolated in 1983 from stools from a human patient in Great Britain. Onthe basis of the biochemical characteristics, this new serovariety wasclassified in the subspecies II but was considered as an atypical strainsince its presence was not detected in gelatinase (Le Minor, L. et al,1984, Supplement No. XXVII, 1983, to Kauffmann-White Scheme, Ann.Microbiol. (Institut Pasteur) 135 B, 45-51). In the light of the resultsreported here, the taxonomic position of the strain 3975-83 ought to bereexamined using the DNA-DNA hybridization technique.

The data presented here indicate that the hybridization method based onthe use of a gene necessary for the invasion of HeLa cells by Salmonellaser. Typhi strain Ty2 can distinguish the Salmonella strains of thesubspecies I from the other enteric bacteria, including E. coli. Thenonradioactive hybridization on a Covalink NH microplate is sensitiveand appropriate for the analysis of a large number of samples.

EXAMPLE 3 DETECTION OF SALMONELLA DNA AMPLIFIED BY SANDWICHHYBRIDIZATION

Sequence of the Oligonucleotides

The DNA fragments chosen are the following (SEQ ID NO: 4-13)(seeposition on the sequence of FIG. 1):

C-terminal part

position

Iag1: 5'-TA TTA AGT ATG CAG GTT ATG-3' 1424-1443

Iag2: 5'-AGA GAA TTT CTG GAA AGT GAA-3' 1585-1605

Iag3: 5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT-3' 1495-1521

Iag4: 5'-GAG CGT GCC TTA CCG ACG ATA-3' 1564-1584

Iag5: 5'-GCA GGG ATC ACT AAG CTG TG-3' 1318-1337

Iag6: 5'-CGT GGG CAA CCA GCA CTA ACG-3' 1637-1657

Slm1: 5'-CG GGT TAA AGG TTA TCA CCT-3' 709-728

Slm2: 5'-AG CAT GGC GCA AAT GGG-3' 1014-1031

Slm3: 5'-GCA CCA GGA AAG CAT TAA GTT GAT AGA ACA C-3' 732-762

Slm4: 5'-CTT CGC TGG GAC ACA AAG CA-3' 823-842

Preferably, the pair of primers Iag5 (sense) and Iag6 (antisense)directs the amplification of a 340 bp fragment, the pair Slm1 (sense)and Slm2 (anti-sense) directs the amplification of a 323 bp fragment(FIG. 3) (SEQ ID NO: 8-11).

FIG. 4 shows the efficiency of the amplification of a pair of primersIag5 and Iag6 on 2 representatives of each of the groups of Salmonellae(SEQ ID NO: 8-9).

Process of Detection

A format for detection by sandwich hybridization was used.

Two oligonucleotides hybridize simultaneously to the denatured amplifiedfragment. One of them, called capture probe, is attached passively (butcan also be attached covalently) to the surface of a 96-well microtitreplate well. The other, called revealing probe, is labelled with anelement which is easy to detect. The revealing probe is free in thehybridization buffer.

The capture and revealing probes are complementary to 2 differentregions situated inside the amplified fragment.

The detection probe in the case described here is linked to an enzymaticmarker, especially a peroxidase, and will serve as revealing probe. Thisis the case preferably for the oligonucleotides Iag3 and Slm3 (SEQ IDNO: 1, 12). Other oligonucleotides can be attached to a microplate-typesolid support, a particulate or membrane support and serve as captureprobe, this particularly for the oligonucleotides Iag4 and Slm4 (SEQ IDNO: 7, 13).

Experimental Conditions

1) Preparation of the Salmonella DNA

Using the boiling method in the presence of Chelex (6% Chelex, 0.1% SDS,1% NP40, 1% Tween 20), the DNA sequences are obtained. This reagent ismarketed by Biorad and is used according to the manufacturer's procedure(ref. Walsh et al. 1991. BioTechniques 10: 506-513).

2) Amplification

According to the method initially described by Saiki and as set out, forexample, in European Patent EP 0,201,184.

The PCR is carried out using the following reaction mixture:

50 mM KCl

10 mM Tris-HCl pH 8.3

1.5 mM MgCl₂

125 μM deoxyribonucleotides (dCTP, dATP, dGTP)

250 μM UTP

25 pmol of each of the primers

10 ng DNA

1 unit of Uracyl N Glycosylase

1 unit of Taq polymerase.

The reaction mixture was prepared using 10 μl of the solution containingthe DNA to be amplified in a volume of 100 μl. The dUTP and UNG are usedin a decontamination system (Brevet Life Technologies European PatentApplication 0 401 037). The thermocycler used is Perkin Elmer 9600.

After incubation at 50° C. for 2 min in order to allow the action of theUNG and denaturation at 95° C. for 5 min, the temperature cycles usedare the following:

5 cycles (95° C. 15 sec, 50° C. 15 sec, 72° C. 15 sec)

35 cycles (95° C. 15 sec, 57° C. 15 sec, 72° C. 15 sec)

3) Visualization of the Amplification Reaction

3-1) Labelling of the Revealing Probe

The probes are labelled with horseradish peroxidase (ref. PCR protocols:a guide to methodes and application; Academic press (1990), 15,p4513-4534) and the activity of the enzyme is revealed by colorimetry.

3-2) Agarose Gel Stained with BET and Membrane Hybridization

After amplification, 10 μl of the amplification product are deposited onan agarose gel and the DNA is transferred onto a membrane according toconventional techniques (Maniatis). The membrane is prehybridized, 30min at 68° C. in hybridization buffer (10 X Denhart, 6 X SSC, 0.1% SDS)and then hybridized at 42° C. for 3 h with 60 ng of probe per ml ofhybridization buffer.

Washing is then carried out according to the following steps:

twice 10 min in 2 X SSC -0.1% SDS at room temperature,

once 30 min in 0.1 X SSC -0.1% SDS at 42° C.,

twice 10 min in 2 X SSC at room temperature.

Revealing: The membrane is blotted between two sheets of absorbent paper(Whatman 3MM paper) and placed in a clean and dry tank.

The Amersham detection reagent (ECL RPN 2105 detection reagent) isprepared immediately before use volume for volume; 30 ml of total volumefor a 5×8 cm membrane. A cassette for autoradiography is obtained byfixing a sheet of absorbent paper (Whatman 3MM paper) at the bottom. Allthese steps can be carried out under light, and then in a dark chamber.

The membrane is immersed in the detection reagent for 1 min, the DNAside on top, the membrane is drained rapidly, it is placed in thecassette, the DNA side on top, a sheet of transparent plastic is placedon top (otherwise the membrane sticks to the film) and an X-ray film isplaced on top (X-OMAT KODAK film).

The exposure is carried out for 30 min at room temperature and then thefilm is developed by conventional developing techniques (developer,water, fixing agent).

3-3) Microplate

3-3-1) Coating of the Capture Oligonucleotide

It can be carried out by adsorption (Cook et al, NAR, 16: 4077-4095(1988) or by covalent coupling (Rasmussen, S. R. et al, 1991. AnalyticalBiochemistry 198, 138-142).

3-3-2) Microplate Hybridization and Reading

10 μl of the amplification product were denatured by adding volume forvolume a 200 mM NaOH, 40 mM EDTA solution.

The microplates in which the surface of the wells is coated with thecapture probe were prehybridized in a hybridization buffer containing 10XDenhart, 6 XSSC, 0.1% SDS.

Next, the microplate was emptied and each of the wells received 200 μlof hybridization buffer containing the denatured amplified fragment andthe revealing probe at the concentration of 10 ng/μl. The incubationtook place for one hour at 37° C. and with stirring.

After washing (10 X washing solution: 100 mM Tris, 3 M NaCl, 1% Tween20, pH 7.4), the activity of the peroxidase linked to the probe wasdetected by colorimetry in the presence of a colored substrate.

To do this, 200 μl of a 40 mM trisodium citrate solution, 0.03% H₂ O30%, 7.5 mg/ml of orthophenylene-diamine (OPD) were distributed in eachof the wells. The microplate was incubated for 30 min in the dark and at37° C. 50 μl/well of a 4 N H₂ SO₄ solution were added in order to blockthe reaction.

The optical density was determined at a wavelength of 492 nm (referenceat 620 nm).

4) Sequencing of the PCR Products and Manual Alignment of the Sequences

According to conventional techniques, by using for example an AppliedBiosystem "373 DNA sequencer" automatic machine and the Applied "dyeterminator" kit.

Results

The exemplified model is preferably the following oligonucleotidesystem:

Iag5 sense primer-Iag6 antisense primer

Iag3 revealing probe and Iag4 capture probe (SEQ ID NO: 8,9,6,7).

(it should be noted that Iag4 can equally well be labelled and used asrevealing probe).

Specificity Study

It was performed on all of the bacterial strains listed in Tables 4 and5.

The amplification of the DNA extracted from the 45 Salmonella strainstested generated a fragment of the expected size (cf. FIG. 5). TheSouthern blots of all the amplified products were hybridized with theinternal oligonucleotide probe Iag3 labelled with peroxidase (SEQ ID NO:6). None of the non-salmonella strains gave rise to hybridization with aperoxidase probe obtained on a membrane according to the proceduredescribed above.

The same amplification products were tested in a mircoplate format.

The cut-off was arbitrarily set at 0.050. All the representatives ofeach of the Salmonella groups give an optical density value greater than0.050 (Table 6).

Sensitivity

In order to determine the minimum number of molecules of salmonellachromosomal DNA which can be detected, a range of dilution of purifiedchromosomal DNA was amplified. 5 molecules are visible on the southernblot autoradiograph and detected by microplate hybridization: the valueobtained by colorimetry is greater than the Cut-Off (FIG. 6).

The oligonucleotides selected for carrying out this example are locatedon the sequence of the IagA gene (FIG. 7).

                  TABLE 4                                                         ______________________________________                                        SALMONELLA STRAINS STUDIED                                                    No.  Strains        Serotype         Group                                    ______________________________________                                        1    Salmonella Marseille            I                                        2    Salmonella Nyanza               I                                        3    Salmonella Poona                I                                        4    Salmonella Kampala              I                                        5    Salmonella Taksony              I                                        6    Salmonella Teshie               I                                        7    Salmonella Indiana              I                                        8    Salmonella enteritidis          I                                        9    Salmonella Kentucky             I                                        10   Salmonella Napoli               I                                        11                  841 11:a:d:en 215                                                                              II                                       12                  1703 K 41:2:15   II                                       13                  950-71 43:d:z 39 II                                       14                  10-65 44:24, 223:-                                                                             II                                       15                  3209-81 45:z 23  II                                       16                  5331/86 62: z 29:-                                                                             IIIa                                     17                  3064-4/252 41:k:-                                                                              IIIa                                     18                  594-54 38:z 54:- IIIa                                     19                  1694 cdai 426 63:z 4, z32:-                                                                    IIIa                                     20                  So 50/16 62:f,z 51:-                                                                           IIIa                                     21                  5251-85 58:r:z 53                                                                              IIIb                                     22                  1758-76 6,14:z 10:enx 215                                                                      IIIb                                     23                  453-68 16:liv:z53                                                                              IIIb                                     24                  4305-57 16:li(v):z 35                                                                          IIIb                                     25                  1698-75 11:liv:z IIIb                                     26                  8275-94 47:r:enx 215                                                                           IIIb                                     27                  8283-94 53:z 10:z                                                                              IIIb                                     28                  cdc 456-5/93 40:i:1,5,7                                                                        IIIb                                     29                  8284-94 60:i:z   IIIb                                     30                  1693 K 38:k:z 55 IIIb                                     31                  1707 48:f:z 51:- IV                                       32                  7231/89 45:z 36, z 38                                                                          IV                                       33                  6887/60 48:f, z 51:-                                                                           IV                                       34                  1357/73 43:z4, z 24:-                                                                          IV                                       35                  1550 K16:z 4, z 23:-                                                                           IV                                       36   Salmonella Bongor                                                                            261-66 48:z35:-  V                                        37   Salmonella Camdeni                                                                           2022-77 44:r:-   V                                        38                  4985-85 48:z 39:-                                                                              V                                        39                  7688-85 48:z39:- V                                        40                  1387-7340:a:-    V                                        41                  1941-77 6,7:z41:1,7                                                                            VI                                       42                  1449 K45:a enx   VI                                       43                  4355-84 1,6,14,25:a:e,n,x                                                                      VI                                       44                  1711 K 11:b:enx  VI                                       45                  1688K,1,6 14,25:Z 10:1,12,7                                                                    VI                                       ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        NON-SALMONELLA STRAINS                                                        No.      Name             Identification                                      ______________________________________                                        1        Klebsiella oxytoca                                                                             0059 SDP                                            2        Klebsiella pneumoniae                                                                          0054 SDP                                            3        Acinetobacter baumanii                                                                         0033 SDP                                            4        Proteus mirabilis                                                                              RP402                                               5        Serratia marcescens                                                                            0042 SDP                                            6        Enterobacter agglomerans                                                                       0067 SDP                                            7        Citrobacter diversus                                                                           0068 SDP                                            8        Pseudomonas aeruginosa                                                                         0011 SDP                                            9        Enterobacter aerogenes                                                                         0066 SDP                                            10       Escherichia coli 0131 SDP                                            11       Enterocoque faecalis                                                                           76117                                               12       Proteus mirabilis                                                                              AP03                                                13       Enterocoque faecalis                                                                           76117                                               14       Enterobacter cloacae                                                                           0060 SDP                                            15       Mycobacterium avium                                                                            6                                                   16       Mycobacterium tuberculosis                                                                     H 37 RV                                             17       Listeria monocytogenes                                                                         1/2 LG3                                             ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        MICROPLATE DETECTION                                                          SAMPLES              OD at 420 nm                                             ______________________________________                                        2 Nyanza gpe I       3.029                                                    3 Poona gpe I        3.103                                                    11 gpe II            3.155                                                    12 gpe II            0.751                                                    18 gpe IIIa          3.139                                                    20 gpe IIIa          3.068                                                    21 gpe IIIb          3.161                                                    30 gpe IIIb          3.201                                                    31 gpe IV            0.272                                                    35 gpe IV            0.527                                                    36 gpe V             1.868                                                    40 gpe V             3.347                                                    45 gpe VI            0.900                                                    Klebsiella oxytoca   0.022                                                    Klebsiella pneumoniae                                                                              0.017                                                    Acinetobacter baumanii                                                                             0.024                                                    Proteus mirabilis    0.019                                                    Serratia marcescens  0.019                                                    Enterobacter agglomerans                                                                           0.023                                                    Mycobacterium avium n° 6                                                                    0.025                                                    Mycobacterium tuberculosis H 37 RV                                                                 0.020                                                    Listeria monocytogenes 1/2 LG3                                                                     0.015                                                    Control water        0.018                                                    Control water        0.022                                                    ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 29                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2406 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (vi) ORIGINAL SOURCE:                                                   #SER. TYPHIA) ORGANISM: SALMONELLA                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 97..1755                                              #/product= "iagA"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1776..2255                                            #/product= "iagB"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GTACTAGCAG CAGAATTACT GAAACAGTAG ATTCTATCCT AACGACTTGT AT - #TAGCTATT         60                                                                          #TTT AAT CCT      114AG AGAATACACT ATTATC ATG CCA CAT                         #    Met Pro His Phe Asn Pro                                                  #   5  1                                                                      - GTT CCT GTA TCG AAT AAA AAA TTC GTC TTT GA - #T GAT TTC ATA CTC AAC          162                                                                          Val Pro Val Ser Asn Lys Lys Phe Val Phe As - #p Asp Phe Ile Leu Asn           #             20                                                              - ATG GAC GGC TCC CTC GTA CGC TCA GAA AAG AA - #A GTC AAT ATT CCG CCA          210                                                                          Met Asp Gly Ser Leu Val Arg Ser Glu Lys Ly - #s Val Asn Ile Pro Pro           #         35                                                                  - AAA GAA TAT GCC GTT CTG GTC ATC CTG CTC GA - #A GCC GCC GGC AAG ATT          258                                                                          Lys Glu Tyr Ala Val Leu Val Ile Leu Leu Gl - #u Ala Ala Gly Lys Ile           #     50                                                                      - GTG AGT AAA AAC ACC TTA TTG GAC CAA GTA TG - #G GGC GAC GCG GAA GTT          306                                                                          Val Ser Lys Asn Thr Leu Leu Asp Gln Val Tr - #p Gly Asp Ala Glu Val           # 70                                                                          - AAC GAA GAA TCT CTT ACC CGC TGT ATC TAT GC - #C TTA CGA CGT ATT CTG          354                                                                          Asn Glu Glu Ser Leu Thr Arg Cys Ile Tyr Al - #a Leu Arg Arg Ile Leu           #                 85                                                          - TCG GAA GAT AAA GAG CAT CGT TAC ATT GAA AC - #A CTG TAC GGA CAG GGT          402                                                                          Ser Glu Asp Lys Glu His Arg Tyr Ile Glu Th - #r Leu Tyr Gly Gln Gly           #            100                                                              - TAT CGG TTT AAT CGT CCG GTC GTA GTG GTG TC - #T CCG CCA GCG CCG CAA          450                                                                          Tyr Arg Phe Asn Arg Pro Val Val Val Val Se - #r Pro Pro Ala Pro Gln           #       115                                                                   - CCT ACG ACT CAT ACA TTG GCG ATA CTT CCT TT - #T CAG ATG CAG GAT CAG          498                                                                          Pro Thr Thr His Thr Leu Ala Ile Leu Pro Ph - #e Gln Met Gln Asp Gln           #   130                                                                       - GTT CAA TCC GAG AGT CTG CAT TAC TCT ATC GT - #G AAG GGA TTA TCG CAG          546                                                                          Val Gln Ser Glu Ser Leu His Tyr Ser Ile Va - #l Lys Gly Leu Ser Gln           135                 1 - #40                 1 - #45                 1 -       #50                                                                           - TAT GCG CCC TTT GGC CTG AGC GTG CTG CCG GT - #G ACC ATT ACG AAG AAC          594                                                                          Tyr Ala Pro Phe Gly Leu Ser Val Leu Pro Va - #l Thr Ile Thr Lys Asn           #               165                                                           - TGC CGC AGT GTT AAG GAT ATT CTT GAG CTC AT - #G GAT CAA TTA CGC CCC          642                                                                          Cys Arg Ser Val Lys Asp Ile Leu Glu Leu Me - #t Asp Gln Leu Arg Pro           #           180                                                               - GAT TAT TAT ATC TCC GGG CAG ATG ATA CCC GA - #T GGT AAT GAT AAT ATT          690                                                                          Asp Tyr Tyr Ile Ser Gly Gln Met Ile Pro As - #p Gly Asn Asp Asn Ile           #       195                                                                   - GTA CAG ATC GAG ATA GTT CGG GTT AAA GGT TA - #T CAC CTG CTG CAC CAG          738                                                                          Val Gln Ile Glu Ile Val Arg Val Lys Gly Ty - #r His Leu Leu His Gln           #   210                                                                       - GAA AGC ATT AAG TTG ATA GAA CAC CAA CCC GC - #T TCT CTC TTG CAA AAC          786                                                                          Glu Ser Ile Lys Leu Ile Glu His Gln Pro Al - #a Ser Leu Leu Gln Asn           215                 2 - #20                 2 - #25                 2 -       #30                                                                           - AAA ATT GCG AAT CTT TTG CTC AGA TGT ATT CC - #C GGA CTT CGC TGG GAC          834                                                                          Lys Ile Ala Asn Leu Leu Leu Arg Cys Ile Pr - #o Gly Leu Arg Trp Asp           #               245                                                           - ACA AAG CAA ATT AGC GAG CTA AAT TCG ATT GA - #C AGT ACC ATG GTC TAC          882                                                                          Thr Lys Gln Ile Ser Glu Leu Asn Ser Ile As - #p Ser Thr Met Val Tyr           #           260                                                               - TTA CGC GGT AAG CAT GAG TTA AAT CAA TAC AC - #C CCC TAT AGC TTA CAG          930                                                                          Leu Arg Gly Lys His Glu Leu Asn Gln Tyr Th - #r Pro Tyr Ser Leu Gln           #       275                                                                   - CAA GCG CTT AAA TTG CTG ACT CAA TGC GTT AA - #T ATG TCG CCA AAC AGC          978                                                                          Gln Ala Leu Lys Leu Leu Thr Gln Cys Val As - #n Met Ser Pro Asn Ser           #   290                                                                       - ATT GCG CCT TAC TGT GCG CTG GCA GAA TGC TA - #C CTC AGC ATG GCG CAA         1026                                                                          Ile Ala Pro Tyr Cys Ala Leu Ala Glu Cys Ty - #r Leu Ser Met Ala Gln           295                 3 - #00                 3 - #05                 3 -       #10                                                                           - ATG GGG ATT TTT GAT AAA CAA AAC GCA ATG AT - #C AAA GCT AAA GAA CAT         1074                                                                          Met Gly Ile Phe Asp Lys Gln Asn Ala Met Il - #e Lys Ala Lys Glu His           #               325                                                           - GCT ATT AAG GCG ACA GAG CTG GAC CAC AAT AA - #T CCA CAA GCT TTA GGA         1122                                                                          Ala Ile Lys Ala Thr Glu Leu Asp His Asn As - #n Pro Gln Ala Leu Gly           #           340                                                               - TTA CTG GGG CTA ATT AAT ACG ATT CAC TCA GA - #A TAC ATC GTC GGG AGT         1170                                                                          Leu Leu Gly Leu Ile Asn Thr Ile His Ser Gl - #u Tyr Ile Val Gly Ser           #       355                                                                   - TTA CTA TTC AAA CAA GCT AAC TTA CTT TCG CC - #C ATT TCT GCA GAT ATT         1218                                                                          Leu Leu Phe Lys Gln Ala Asn Leu Leu Ser Pr - #o Ile Ser Ala Asp Ile           #   370                                                                       - AAA TAT TAT TAT GGC TGG AAT CTT TTC ATG GC - #T GGT CAG TTG GAG GAG         1266                                                                          Lys Tyr Tyr Tyr Gly Trp Asn Leu Phe Met Al - #a Gly Gln Leu Glu Glu           375                 3 - #80                 3 - #85                 3 -       #90                                                                           - GCC TTA CAA ACG ATT AAC GAG TGT TTA AAA TT - #G GAC CCA ACG CGC GCA         1314                                                                          Ala Leu Gln Thr Ile Asn Glu Cys Leu Lys Le - #u Asp Pro Thr Arg Ala           #               405                                                           - GCC GCA GGG ATC ACT AAG CTG TGG ATT ACC TA - #T TAT CAT ACC GGT ATT         1362                                                                          Ala Ala Gly Ile Thr Lys Leu Trp Ile Thr Ty - #r Tyr His Thr Gly Ile           #           420                                                               - GAT GAT GCT ATA CGT TTA GGC GAT GAA TTA CG - #C TCA CAA CAC CTG CAG         1410                                                                          Asp Asp Ala Ile Arg Leu Gly Asp Glu Leu Ar - #g Ser Gln His Leu Gln           #       435                                                                   - GAT AAT CCA ATA TTA TTA AGT ATG CAG GTT AT - #G TTT CTT TCG CTT AAA         1458                                                                          Asp Asn Pro Ile Leu Leu Ser Met Gln Val Me - #t Phe Leu Ser Leu Lys           #   450                                                                       - GGT AAA CAT GAA CTG GCA CGA AAA TTA ACT AA - #A GAA ATA TCC ACG CAG         1506                                                                          Gly Lys His Glu Leu Ala Arg Lys Leu Thr Ly - #s Glu Ile Ser Thr Gln           455                 4 - #60                 4 - #65                 4 -       #70                                                                           - GAA ATA ACA GGA CTT ATT GCT GTT AAT CTT CT - #T TAC GCT GAA TAT TGT         1554                                                                          Glu Ile Thr Gly Leu Ile Ala Val Asn Leu Le - #u Tyr Ala Glu Tyr Cys           #               485                                                           - CAG AAT AGT GAG CGT GCC TTA CCG ACG ATA AG - #A GAA TTT CTG GAA AGT         1602                                                                          Gln Asn Ser Glu Arg Ala Leu Pro Thr Ile Ar - #g Glu Phe Leu Glu Ser           #           500                                                               - GAA CAG CGT ATA GAT AAT AAT CCG GGA TTA TT - #A CCG TTA GTG CTG GTT         1650                                                                          Glu Gln Arg Ile Asp Asn Asn Pro Gly Leu Le - #u Pro Leu Val Leu Val           #       515                                                                   - GCC CAC GGC GAA GCT ATT GCC GAG AAA ATG TG - #G AAT AAA TTT AAA AAC         1698                                                                          Ala His Gly Glu Ala Ile Ala Glu Lys Met Tr - #p Asn Lys Phe Lys Asn           #   530                                                                       - GAA GAC AAT ATT TGG TTC AAA AGA TGG AAA CA - #G GAT CCC CGC TTG ATT         1746                                                                          Glu Asp Asn Ile Trp Phe Lys Arg Trp Lys Gl - #n Asp Pro Arg Leu Ile           535                 5 - #40                 5 - #45                 5 -       #50                                                                           - AAA TTA CGG TAAAATCTGA GAGAGGAGAT ATG CAT TAT TT - #T TTT ATC ATC           1796                                                                          #Ile Ile  Met His Tyr Phe Phe                                                 #1               5                                                            - GTA ATC TGG TTG CTT AGC ATA AAT ACG GCA TG - #G GCT GAT TGC TGG CTT         1844                                                                          Val Ile Trp Leu Leu Ser Ile Asn Thr Ala Tr - #p Ala Asp Cys Trp Leu           #         20                                                                  - CAG GCT GAA AAA ATG TTC AAT ATT GAA TCC GA - #A CTA CTT TAC GCT ATC         1892                                                                          Gln Ala Glu Lys Met Phe Asn Ile Glu Ser Gl - #u Leu Leu Tyr Ala Ile           #     35                                                                      - GCC CAG CAG GAA TCG GCG ATG AAA CCT GGC GC - #C ATT GGT CAT AAC CGA         1940                                                                          Ala Gln Gln Glu Ser Ala Met Lys Pro Gly Al - #a Ile Gly His Asn Arg           # 55                                                                          - GAT GGT TCA ACC GAT CTT GGC CTG ATG CAA AT - #T AAC AGC TTC CAT ATG         1988                                                                          Asp Gly Ser Thr Asp Leu Gly Leu Met Gln Il - #e Asn Ser Phe His Met           #                 70                                                          - AAA AGG CTG AAA AAA ATG GGG ATT AGT GAA AA - #A CAG TTG TTA CAG GAT         2036                                                                          Lys Arg Leu Lys Lys Met Gly Ile Ser Glu Ly - #s Gln Leu Leu Gln Asp           #             85                                                              - CCC TCG ATT TCT GTC ATT GTG GGC GCA TCC AT - #T TTA TCA GAT ATG ATG         2084                                                                          Pro Ser Ile Ser Val Ile Val Gly Ala Ser Il - #e Leu Ser Asp Met Met           #        100                                                                  - AAA ATC TAC GGT TTT AGC TGG GAG GCC GTT GG - #C GCT TAT AAT GCC GGG         2132                                                                          Lys Ile Tyr Gly Phe Ser Trp Glu Ala Val Gl - #y Ala Tyr Asn Ala Gly           #   115                                                                       - ACG TCG CCG AAA CGA TCG GAT ATA AGG AAA CG - #T TAT GCT AAA AAA ATT         2180                                                                          Thr Ser Pro Lys Arg Ser Asp Ile Arg Lys Ar - #g Tyr Ala Lys Lys Ile           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - TGG GAG AAT TAC AGA AAA TTA AAA GAG ATG TC - #A GCA GAA GAG AAA AAC         2228                                                                          Trp Glu Asn Tyr Arg Lys Leu Lys Glu Met Se - #r Ala Glu Glu Lys Asn           #               150                                                           - AAA AGA CTT TCT ATC GCG GTA AAC AAA TAATTATAC - #A GGAATAGCTT               2275                                                                          Lys Arg Leu Ser Ile Ala Val Asn Lys                                           #           160                                                               - ACTTTCAGAT AATTCTAAAA GTAAGCTATG TTTTTATCAG CTTGCCGTCG TC - #ATAAGCAA       2335                                                                          - CTGGCGCTTG CATTGCTTTT AGTTGTACAA ACTGTGAGGC GTCTTCCAGC AT - #TCTATTGT       2395                                                                          #     2406                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 553 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Pro His Phe Asn Pro Val Pro Val Ser As - #n Lys Lys Phe Val Phe         #                 15                                                          - Asp Asp Phe Ile Leu Asn Met Asp Gly Ser Le - #u Val Arg Ser Glu Lys         #             30                                                              - Lys Val Asn Ile Pro Pro Lys Glu Tyr Ala Va - #l Leu Val Ile Leu Leu         #         45                                                                  - Glu Ala Ala Gly Lys Ile Val Ser Lys Asn Th - #r Leu Leu Asp Gln Val         #     60                                                                      - Trp Gly Asp Ala Glu Val Asn Glu Glu Ser Le - #u Thr Arg Cys Ile Tyr         # 80                                                                          - Ala Leu Arg Arg Ile Leu Ser Glu Asp Lys Gl - #u His Arg Tyr Ile Glu         #                 95                                                          - Thr Leu Tyr Gly Gln Gly Tyr Arg Phe Asn Ar - #g Pro Val Val Val Val         #           110                                                               - Ser Pro Pro Ala Pro Gln Pro Thr Thr His Th - #r Leu Ala Ile Leu Pro         #       125                                                                   - Phe Gln Met Gln Asp Gln Val Gln Ser Glu Se - #r Leu His Tyr Ser Ile         #   140                                                                       - Val Lys Gly Leu Ser Gln Tyr Ala Pro Phe Gl - #y Leu Ser Val Leu Pro         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Thr Ile Thr Lys Asn Cys Arg Ser Val Ly - #s Asp Ile Leu Glu Leu         #               175                                                           - Met Asp Gln Leu Arg Pro Asp Tyr Tyr Ile Se - #r Gly Gln Met Ile Pro         #           190                                                               - Asp Gly Asn Asp Asn Ile Val Gln Ile Glu Il - #e Val Arg Val Lys Gly         #       205                                                                   - Tyr His Leu Leu His Gln Glu Ser Ile Lys Le - #u Ile Glu His Gln Pro         #   220                                                                       - Ala Ser Leu Leu Gln Asn Lys Ile Ala Asn Le - #u Leu Leu Arg Cys Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Leu Arg Trp Asp Thr Lys Gln Ile Se - #r Glu Leu Asn Ser Ile         #               255                                                           - Asp Ser Thr Met Val Tyr Leu Arg Gly Lys Hi - #s Glu Leu Asn Gln Tyr         #           270                                                               - Thr Pro Tyr Ser Leu Gln Gln Ala Leu Lys Le - #u Leu Thr Gln Cys Val         #       285                                                                   - Asn Met Ser Pro Asn Ser Ile Ala Pro Tyr Cy - #s Ala Leu Ala Glu Cys         #   300                                                                       - Tyr Leu Ser Met Ala Gln Met Gly Ile Phe As - #p Lys Gln Asn Ala Met         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Lys Ala Lys Glu His Ala Ile Lys Ala Th - #r Glu Leu Asp His Asn         #               335                                                           - Asn Pro Gln Ala Leu Gly Leu Leu Gly Leu Il - #e Asn Thr Ile His Ser         #           350                                                               - Glu Tyr Ile Val Gly Ser Leu Leu Phe Lys Gl - #n Ala Asn Leu Leu Ser         #       365                                                                   - Pro Ile Ser Ala Asp Ile Lys Tyr Tyr Tyr Gl - #y Trp Asn Leu Phe Met         #   380                                                                       - Ala Gly Gln Leu Glu Glu Ala Leu Gln Thr Il - #e Asn Glu Cys Leu Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Asp Pro Thr Arg Ala Ala Ala Gly Ile Th - #r Lys Leu Trp Ile Thr         #               415                                                           - Tyr Tyr His Thr Gly Ile Asp Asp Ala Ile Ar - #g Leu Gly Asp Glu Leu         #           430                                                               - Arg Ser Gln His Leu Gln Asp Asn Pro Ile Le - #u Leu Ser Met Gln Val         #       445                                                                   - Met Phe Leu Ser Leu Lys Gly Lys His Glu Le - #u Ala Arg Lys Leu Thr         #   460                                                                       - Lys Glu Ile Ser Thr Gln Glu Ile Thr Gly Le - #u Ile Ala Val Asn Leu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Ala Glu Tyr Cys Gln Asn Ser Glu Ar - #g Ala Leu Pro Thr Ile         #               495                                                           - Arg Glu Phe Leu Glu Ser Glu Gln Arg Ile As - #p Asn Asn Pro Gly Leu         #           510                                                               - Leu Pro Leu Val Leu Val Ala His Gly Glu Al - #a Ile Ala Glu Lys Met         #       525                                                                   - Trp Asn Lys Phe Lys Asn Glu Asp Asn Ile Tr - #p Phe Lys Arg Trp Lys         #   540                                                                       - Gln Asp Pro Arg Leu Ile Lys Leu Arg                                         545                 5 - #50                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 160 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met His Tyr Phe Phe Ile Ile Val Ile Trp Le - #u Leu Ser Ile Asn Thr         #                 15                                                          - Ala Trp Ala Asp Cys Trp Leu Gln Ala Glu Ly - #s Met Phe Asn Ile Glu         #             30                                                              - Ser Glu Leu Leu Tyr Ala Ile Ala Gln Gln Gl - #u Ser Ala Met Lys Pro         #         45                                                                  - Gly Ala Ile Gly His Asn Arg Asp Gly Ser Th - #r Asp Leu Gly Leu Met         #     60                                                                      - Gln Ile Asn Ser Phe His Met Lys Arg Leu Ly - #s Lys Met Gly Ile Ser         # 80                                                                          - Glu Lys Gln Leu Leu Gln Asp Pro Ser Ile Se - #r Val Ile Val Gly Ala         #                 95                                                          - Ser Ile Leu Ser Asp Met Met Lys Ile Tyr Gl - #y Phe Ser Trp Glu Ala         #           110                                                               - Val Gly Ala Tyr Asn Ala Gly Thr Ser Pro Ly - #s Arg Ser Asp Ile Arg         #       125                                                                   - Lys Arg Tyr Ala Lys Lys Ile Trp Glu Asn Ty - #r Arg Lys Leu Lys Glu         #   140                                                                       - Met Ser Ala Glu Glu Lys Asn Lys Arg Leu Se - #r Ile Ala Val Asn Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 # 20               TATG                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #21                GTGA A                                                     - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #             27   TAAC AGGACTT                                               - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #21                CGAT A                                                     - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 # 20               TGTG                                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #21                TAAC G                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                # 20               ACCT                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #          31      AAGT TGATAGAACA C                                          - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                # 20               AGCA                                                       - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                # 20               TGCT                                                       - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #                24CAGC AGGA                                                  - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #            29    GGTT CAAACGATC                                             - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                # 20               CCCT                                                       - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - TATTATCATA CCGGTATTGA TGATGCTATA CGTTTAGGCG ATGAATTACG CT - #CACAACAC         60                                                                          - CTGCAGGATA ATCCAATATT ATTAAGTATG CAGGTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAACTGG CACGAAAATT AACTAAAGAA ATATCCACGC AGGAAATAAC AG - #GACTTATT        180                                                                          - GCTGTTAATC TTCTTTACGC TGAATATTGT CAGAATAGTG AGCGTGCCTT AC - #CGACGATA        240                                                                          - AGAGAATTTC TGGAAAGTGA ACAGCGTATA GATAATAATC CGGGATTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - TATTATCATA CTGGTATTGA TGATGCTATA CGTTTAGGCG ATGAATTACG CT - #CACAACAC         60                                                                          - CTGCAGGATA ATCCAATATT ATTAAGTATG CAGGTTATGT TTCTTTCTCT TA - #AAGGTAAA        120                                                                          - CATGAACTGG CACGAAAATT ATCTAAAGAA ATATCCACGC AGGAAATAAC AG - #GGCTTATT        180                                                                          - GCTGTTAATC TTCTTTATGC TGAATACTGT CAGAATAGTG AGCGTGCCTT AC - #CGACGATA        240                                                                          - AGAGAATTTC TGGAAAGTGA ACAGCGTATA GATAATAATC CGGGATTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - TATTATCATA CCGGCATTGA TGATGCCATA CGTTTAGGAG ATGAACTACG CT - #CACAGCAC         60                                                                          - CTGCAGGATA ATCCCATTTT ATTAAGTATG CAGGTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAGCTGG CACGAAAATT AACTAAAGAG ACATCCCCGC ATGAGATAAC AG - #GGCTTATT        180                                                                          - GCTATTAATC TTCTTTATGC TGAATACTGT CAGAATAGTG AGCGAGCCTT AC - #CGAGGATA        240                                                                          - AGAGAATATC TGGCAAGTGA ACGGAGTATT GATAATAATC CTGGACTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - TATTATCATG CCGGCATTGA TGATGCTATA CGTTTAGGAG ATGAATTACG TT - #CACAACAT         60                                                                          - CTGCAGGATA ATCCAATACT ATTAAGTATG CAGGTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAACTGG CACGAAAATT AGCTAAAGAA ATATCCAAGC ATGAAATAAC AG - #GGCTTATT        180                                                                          - GCTGTTAATC TTCTGTATGC TGAATACTGT CAGAATAGCG AGCGTGCATT AC - #CGAGGATA        240                                                                          - AGAGAGTTTC TGGAAAGTGA ACAGAATACT GATAATAATC CCGGGCTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - TACTATCATA CTGGCCTTGA TGATGCTATA CGTTTAGGAG ATGAATTACG TT - #CGCAACAT         60                                                                          - TTGCAGGATA ATCCAATATT ATTAAGTATG CAGGTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAACTGG CACGAAAATT AACTAAAGAA ATATCCACAC ATGAAATAAC AG - #GGCTTATT        180                                                                          - GCTGTTAATC TTCTTTATGC TGAATACTGT CAGAATAGTG AGCGTGCCTT AG - #CGACGATA        240                                                                          - AGAGAATTTC TGGAAAGTGA ACAGAGTGTT GATAATAACC CAGGGTTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - TATTATCAGA CTGGCATTGA TGATGCTATA CGTTTAGGCG ATGAATTACG CT - #CACAATAT         60                                                                          - CTGCAAGATA ATCCAATATT ATTAAGTATG CAGCTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAGTTGG CACGAAAATT AGCTAAAGAA ATATCCACAC ACGAAGTAAC AG - #GGCTTATT        180                                                                          - GCTGTTAATC TTCTTTATGC TGAATACTGT CAGAATAGCG AGCGTGCTTT AC - #CGGCGATA        240                                                                          - AGAGAATTTC TGGAAAGTGA ACAGAATATA GATAATAATC CGGGGCTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - TACTATCATA CAGGCCTTGA TGATGCTATA CGTTTAGGCG ATGAATTACG TA - #CACAACAT         60                                                                          - TTGCAAGATA ATCCTATATT ATTAAGTATG CAAGTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAACTGG CACGGCTATT AGCTAAAGAA ATATCCTCAC ATGAAATAAC AG - #GGCTTATT        180                                                                          - GCTATTAATC TTCTTTATGC TGAATATTGT CAGAATAGTG AGCGCGCCTT AC - #CGGCGATA        240                                                                          - AAAGAATTTC TGGAAAGTGA ACAAAATATT GACAATAACC CTGGGCTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 300 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - TATTATCATA CCGGCATTGA TGATGCTATA CGGTTAGGAG ATGAATTACG TT - #CACAACAC         60                                                                          - TTGCAGGATA ATCCAATATT ATTAAGTATG CAGGTTATGT TTCTTTCGCT TA - #AAGGTAAA        120                                                                          - CATGAACTGG CACGAAAATT AACTAAAGAA ATATCCAGAC ATGAAATAAC AG - #GGCTTATT        180                                                                          - GCTGTTAATC TTCTTTATGC TGAATACTGT CAGAATAGTG AGCGTGCCTT AT - #CGAGGATA        240                                                                          - AGAGAATTTC TGGAAAGTGA ACAGAGTATT GATAATAATC CAGGACTATT AC - #CGTTAGTG        300                                                                          - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                #        34        CTGT ACAATATTAT CATT                                       - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #  18              AA                                                         __________________________________________________________________________

What is claimed is:
 1. An isolated DNA sequence consisting of at most2406 nucleotides and comprising the nucleotide sequence of SEQ ID NO: 1.2. An isolated DNA sequence consisting of at most 2406 nucleotides whichcomprises a nucleotide sequence which encodes the protein of SEQ ID NO:2 or
 3. 3. The DNA of claim 2 comprising the nucleotide sequence frombase 97 to 1755 of SEQ ID NO:
 1. 4. The DNA of claim 2 comprising thenucleotide sequence from base 1776 to 2255 of SEQ ID NO:
 1. 5. Anisolated and purified protein encoded by the DNA of claim
 2. 6. Anisolated and purified protein encoded by the DNA of claim 3 andcomprising the amino acid sequence of SEQ ID NO:
 2. 7. An isolated andpurified protein encoded by the DNA of claim 4 comprising the amino acidsequence of SEQ ID NO:
 3. 8. A method of producing amplified DNA,comprisingcontacting a DNA or cDNA sequence of SEQ ID NO: 1 with a DNAsequence consisting of at most 34 nucleotides in length and comprising anucleotide sequence selected from the group consisting of SEQ ID NO: 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 28 and 29, andamplifying said DNA or cDNA sequence.
 9. The method of claim 8, furthercomprising detecting the amplified DNA or cDNA sequence with anoligonucleotide having the sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 28 or
 29. 10. A method of producingamplified DNA, comprising:contacting DNA or cDNA from a bacterium of thespecies S. enterica and/or S. bongori of group I, II, III, IV or V withthe pair of DNA sequences of SEQ ID NO: 8 and 9 or SEQ ID NO: 10 and 11,and amplifying said DNA or cDNA.
 11. A method of detecting DNA,comprising:(a) denaturing a sample of amplified S. enterica DNA; (b)contacting denatured S. enterica DNA from step (a) with a labeledrevealing probe comprising the sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 28 or 29, and a capture probehaving the sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 28 or 29, in a hybridization buffer for a timesufficient to allow said revealing probe and said capture probe tohybridize with said denatured S. enterica DNA;wherein said capture probeis attached to the surface of a well of a microtiter plate; (c) washingwith an aqueous buffer to remove unhybridized revealing probe; and (d)detecting said revealing probe which is hybridized to said denatured S.enterica DNA.
 12. The method of claim 11, wherein said revealing probecomprising the sequence of SEQ ID NO: 6 and said capture probe comprisesthe sequence of SEQ ID NO:
 7. 13. The method of claim 11, wherein saiddenaturing step (a) comprises combining 10 μL of an aqueous solutioncontaining therein said amplified S. enterica DNA with 10 μL of anaqueous solution containing therein 200 mM NaOH and 40 mMethylenediaminetetraacetic acid.
 14. The method of claim 11, whereinsaid revealing probe comprises the sequence of SEQ ID NO: 6 and islabeled with peroxidase and said capture probe comprises the sequence ofSEQ ID NO:
 7. 15. The method of claim 11, wherein said time sufficientto allow said revealing probe and said capture probe to hybridize withsaid denatured S. enterica DNA is one hour at 37° C.
 16. The method ofclaim 11, wherein said aqueous buffer in step (c) is an aqueous solutioncontaining therein 100 mM Tris-HCl and 3 M NaCl, pH 7.4.
 17. The methodof claim 16, wherein said revealing probe is labeled with peroxidase andsaid detecting step (d) comprises measuring the activity of saidperoxidase label with a colored substrate.
 18. The method of claim 17,wherein said detecting step comprises:(d₁) adding 200 μL of an aqueoussolution containing 40 mM trisodium citrate, 0.03% 30% H₂ O₂, and 7.5mg/ml of ortho-phenylenediamine to said well of said microtiter plate;(d₂) incubating said microtiter plate for 30 minutes in the dark at 37°C.; (d₃) adding 50 μL of an aqueous solution containing therein 4 N H₂SO₄ ; and (d₄) determining the optical density at a wavelength of 492nm.
 19. A purified and/or isolated protein consisting of SEQ ID NO: 2.20. A purified and/or isolated protein consisting of SEQ ID NO: 3or afragment thereof, wherein said fragment is specifically recognized by anantibody capable of specifically recognizing an epitope of a proteinconsisting of SEQ ID NO:
 3. 21. The protein of claim 20, wherein saidfragment allows a bacteria of the species S. typhi to adhere and infectHeLa cells in culture.
 22. A DNA sequence consisting of at most 2406nucleotides which comprises a nucleotide sequence which encodes theprotein of claim
 20. 23. A DNA sequence consisting of at most 2406nucleotides which comprises a nucleotide sequence which encodes theprotein of claim
 21. 24. A DNA consisting of the nucleotide sequence ofSEQ ID NO:
 1. 25. The DNA of claim 3, consisting of the nucleotidesequence from base 97 to 1755 of SEQ ID NO:
 1. 26. The DNA of claim 4,consisting of the nucleotide sequence from base 1776 to 2255 of SEQ IDNO: 1.